Novel protein and use thereof

ABSTRACT

A novel gene likely inhibiting the onset and progress of cancer. A protein having an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:4 or its salt; a polynucleotide encoding the same; and medicinal use, etc. thereof are provided.

TECHNICAL FIELD

This invention relates to a novel protein and use thereof

BACKGROUND ART

For the functional actions of cells, it is essential that individualcells constituting many living organisms including human beingsrecognize intracellular environments accurately to cope rapidlytherewith. For this purpose, various molecules present on the surface ofcells have important functions, and in particular many protein moleculesincluding receptors play the central role. For example, many Tyrkinase-type receptors corresponding to extracellular ligands function byphosphorylating specific intracellular proteins. Further, some surfaceproteins expressed in a certain kind of leukocytes participate inimmunoreaction in the body by acting on other cells throughintercellular interaction. Many of these protein molecules are presenton and fixed in some forms to the cell membrane. However, some arereleased extracellularly by an enzyme or via a certain other mechanism,to function as secretory proteins. There are diverse secretory proteinsincluding those enhancing or antagonizing original functions or thosehaving completely different functions. For example, a large number ofsecretory receptors compete for ligands with receptors on the membrane.Further, molecules such as CD55 and CD59, whether secretory type ormembrane-bound type, work for repression of the complement system(Immunology Today 20, 576-582 (1999)).

On one hand, a large number of genes for proteins such as cell growthfactors or their receptors, transcription factors, and proteins involvedin signal transduction are found as cancer-related genes. For example,there are PDGF relating to brain tumor (Nature 362, 801 (1997)), c-mycand N-myc relating to breast cancer, stomach cancer and neuroblastoma,and Ki-ras and N-ras relating to colon cancer and leukemia. These geneswere identified from information on the sequences of specific proteinsand genes found upon transformation of specific cells and tissues, andare not commonly expressed on all or many cancer cells or tissues.Further, the immune systems for mammals including humans are verycomplicated, and the elucidation of the mechanism of development andprogress of all cancers by a single gene product is expected to bedifficult.

Accordingly, cancer-related genes involved in cancer development andprogress or genes coding for a group of proteins called cancer antigenmolecules whose expression is enhanced upon transformation attractattention in recent years, and there is need for discovery of a new genewhich may directly or indirectly promote cancer development and progressby particularly participating in some way in the immune system.

SUMMARY OF INVENTION

The present inventors made extensive study to solve the problemdescribed above, and as a result, they found a novel gene coding for amembrane-bound and/or secretory protein having a hydrophobic amino acidcluster in both N- and C-terminals on a predicted amino acid sequence.

On the basis of these findings, the present inventors made furtherstudy, to complete the present invention.

That is, the present invention provides:

1. A protein comprising an amino acid sequence identical orsubstantially identical with the amino acid sequence set forth in SEQ IDNO:4 or a salt thereof,

2. A partial peptide of the protein described in item 1, or an amide, anester or a salt thereof,

3. A DNA comprising a DNA coding for the protein described in item 1;

4. The DNA according to item 3, which comprises the nucleotide sequenceset forth in SEQ ID NO:3,

5. A DNA comprising a DNA coding for the partial peptide described initem 2,

6. A recombinant vector comprising the DNA described in item 3 or 5;

7. A transformant transformed with the recombinant vector described initem 6,

8. A process for producing the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof, which comprises culturing the transformantdescribed in item 7, and obtaining the protein of item 1, or the partialpeptide of item 2,

9. An antibody against the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof,

10. A diagnostic composition comprising the DNA described in item 3 or5, or the antibody described in item 9,

11. A pharmaceutical composition comprising the protein described initem 1 or a salt thereof, or the partial peptide described in item 2 oran amide, an ester or a salt thereof, or the antibody described in claim9,

12. A pharmaceutical composition according to item 11, for preventingand treating cancers,

13. A method of screening a compound or a salt thereof promoting orinhibiting an activity of the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof, which comprises using the protein described initem 1 or a salt thereof, or the partial peptide described in item 2 oran amide thereof, an ester thereof or a salt thereof,

14. A kit for screening a compound or a salt thereof promoting orinhibiting an activity of the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof, which comprises the protein described in item 1or a salt thereof, or the partial peptide described in item 2 or anamide thereof, an ester thereof or a salt thereof,

15. A compound or a salt thereof promoting an activity of the proteindescribed in item 1 or a salt thereof, or the partial peptide describedin item 2 or an amide, an ester or a salt thereof, which is obtainableby the screening method described in item 13 or the screening kitdescribed in item 14,

16. A pharmaceutical composition comprising the compound described initem 15 or a salt thereof,

17. The pharmaceutical composition according to item 16, for preventingand treating cancer,

18. Use of (i) the protein described in item 1 or a salt thereof, or thepartial peptide described in item 2 or an amide, an ester or a saltthereof, or (ii) the compound or a salt thereof promoting an activity ofthe protein described in item 1 or a salt thereof, or the partialpeptide described in item 2 or an amide, an ester or a salt thereof,which is obtained by the screening method described in item 13 or thescreening kit described in item 14, for producing a pharmaceuticalcomposition having an anticancer action,

19. A method of preventing and treating cancer, which comprisesadministering to mammals (i) the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof, or (ii) the compound or a salt thereofpromoting an activity of the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof, which is obtained by the screening methoddescribed in item 13 or the screening kit described in item 14.

20. A compound or a salt thereof inhibiting an activity of the proteindescribed in item 1 or a salt thereof, or the partial peptide describedin item 2 or an amide, an ester or a salt thereof, which is obtained bythe screening method described in item 13 or the screening kit describedin item 14,

21. A pharmaceutical composition comprising the compound described initem 20 or a salt thereof,

22. The pharmaceutical composition according to item 21, which is animmunosupressant or an antiinflammatory composition,

23. Use of (i) the compound or a salt thereof inhibiting the activity ofthe protein described in item 1 or a salt thereof, or the partialpeptide described in item 2 or an amide, an ester or a salt thereof,which is obtained by the screening method described in item 13 or thescreening kit described in item 14, or (ii) the antibody described initem 9, for producing a pharmaceutical composition having animmunosuppressive or antiinflammatory action, and

24. A method of immunorepression or a method of treating inflammations,which comprises administering to mammals the compound or a salt thereofinhibiting the activity of the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide thereof,an ester thereof or a salt thereof, which is obtained by the screeningmethod described in item 13 or the screening kit described in item 14,or the antibody described in item 9.

Further, this invention provides:

25. The protein according to item 1 or a salt thereof, wherein the aminoacid sequence substantially identical with the amino acid sequence setforth in SEQ ID NO:4 is an amino acid sequence having about 70% or more,preferably about 80% or more, more preferably about 90% or more, stillmore preferably about 95% or more homology with the amino acid sequenceset forth in SEQ ID NO:4,

26. The protein according to item 1 or a salt thereof, wherein the aminoacid sequence substantially identical with the amino acid sequence setforth in SEQ ID NO:4 is (i) an amino acid sequence set forth in SEQ IDNO:4 wherein 1 to 5 (preferably 1 to 3) amino acids are deleted, (ii) anamino acid sequence set forth in SEQ ID NO:4 wherein 1 to 10 (preferably1 to 5 (more preferably 1 to 3)) amino acids are added, (iii) an aminoacid sequence set forth in SEQ ID NO:4 wherein 1 to 5 (preferably 1 to3) amino acids are replaced by other amino acids, or (iv) a combinationof the above amino acid sequences,

27. The screening method described in item 13, which comprises measuringan activity of the protein described in item 1 or a salt thereof, or thepartial peptide described in item 2 or an amide, an ester or a saltthereof, and comparing the activity between in the case (i) where asubstrate is contacted with the protein described in item 1 or a saltthereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof and in the case (ii) where the substrate and atest compound are contacted with the protein described in item 1 or asalt thereof, or the partial peptide described in item 2 or an amide, anester or a salt thereof,

28. A method of quantifying the protein described in item 1, or thepartial peptide described in item 2 or a salt thereof in a testsolution, which comprises competitively reacting a test solution and thelabeled protein described in item 1 or a salt thereof, or the labeledpartial peptide described in item 2 or an amide, an ester or a saltthereof with the antibody described in item 9, and then measuring aproportion of the labeled protein described in item 1 or a salt thereof,or the labeled partial peptide described in item 2 or an amide, an esteror a salt thereof which is bound to the antibody, and

29. A method of quantifying the protein described in item 1, or thepartial peptide described in item 2 or a salt thereof in a testsolution, which comprises reacting a test solution simultaneously orsuccessively with the antibody of item 9 insolubilized on a carrier andthe labeled antibody of item 9, and then measuring an activity of thelabel on the insolubilizing carrier.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a Western blotting showing expression of CSP-FLAG in COS-7cells.

FIG. 2A is a histogram of FITC when a concentrate of a supernatantculture of CSP2-Fc-expressing COS7 cells was added to CHO-K1/hNKG2D-11cells; B is a histogram of FITC when a concentrate of a supernatantculture of CSP2-Fc-expressing COS7 cells obtained in Example 6 was addedto CHO-K1 cells (control); C is a histogram of FITC when a concentrateof a supernatant culture of a Mock plasmid-containing COS7 cells wasadded to CHO-K1/hNKG2D-11 cells; and D is a combination of histograms Ato C.

BEST MODE OF THE INVENTION

The protein having an amino acid sequence identical or substantiallyidentical with the amino acid sequence set forth in SEQ ID NO:4 in thepresent invention (referred to hereinafter as the protein of theinvention) may be a protein derived from cells (e.g., hepatic cells,spleen cells, nerve cells, glia cells, pancreatic β-cells, bone marrowcells, mesangial cells, Langerhans cells, epidermal cells, epithelialcells, endothelial cells, fibroblasts, fibrous cells, muscular cells,adipocytes, immunocytes (e.g., macrophage, T cells, B cells, naturalkiller cells, mast cells, neutrophils, basophils, acidophils,monocytes), megakaryocytes, synovial membrane cells, cartilage cells,bone cells, osteoblasts, osteoclasts, mammary gland cells, hepatic cellsor interstitial cells, or their precursor cells, stem cells or cancercells) or any tissues having such cells, for example, the brain and eachsite of the brain (e.g., olfactory bulb, tonsil nuclei, cerebrum basalganglion, hippocampus, thalamus, hypothalamus, cerebral cortex, medullabulb, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney,liver, gonads, thyroid glands, galls, bone marrow, adrenals, skin,muscles, lungs, digestive tracts (e.g., large and small intestines),blood vessels, heart, thymus, spleen, salivary glands, peripheral blood,prostate, testicles, ovary, placenta, uterus, bone, joints, skeletalmuscles, or corpuscle cells or cultured cells thereof (for example, MEL,M1, CTLL-2, HT-2, WEHI-3, HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4,MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78,HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01 etc.) fromhumans or warm-blooded animals (e.g., guinea pig, rat, mouse, chicken,rabbit, pig, sheep, cow, monkey etc.), as well as a synthetic protein.

The amino acid sequence substantially identical with the amino acidsequence set forth in SEQ ID NO:4 includes those amino acid sequenceshaving about 70% or more, preferably about 80% or more, more preferablyabout 90% or more, still more preferably about 95% or more homology withthe amino acid sequence set forth in SEQ ID NO:4.

The protein of the invention having an amino acid sequence substantiallyidentical with the amino acid sequence set forth in SEQ ID NO:4 ispreferably e.g. a protein having an amino acid sequence substantiallyidentical with the amino acid sequence set forth in SEQ ID NO:4 andhaving substantially identical properties with those of a protein havingthe amino acid sequence set forth in SEQ ID NO:4.

“The substantially identical properties” means, for example, theactivity of binding to NKG2D and the activation of immunocytes.

The term “substantially identical” means that between those proteins inquestion, their properties are identical qualitatively (e.g.physiochemically or pharmacologically). It follows that between thoseproteins in question, their qualitative properties such as activity ofbinding to NKG2D and activation of immunocytes are preferably identical(e.g., about 0.1- to 100-fold, preferably about 0.5- to 10-fold, morepreferably about 0.5- to 2-fold), while their quantitative propertiessuch as degree of these activities and molecular weight may bedifferent.

The activity of binding to NKG2D may be measured by a method known perse, for example ELISA etc. Alternatively, the activity may also bemeasured according to a screening method described below.

Measurement of the activation of immunocytes can be carried outaccording to a method known per se, and for example, there is a methodwherein growth of immunocytes is examined as an indicator of theactivation of immunocytes. Specifically, this method comprises measuringintracellular DNA synthesis, where DNA synthesis can usually bequantified in terms of incorporation of thymidine or its derivative.That is, there is a method wherein incorporation of [³H]thymidine isquantified in terms of its radioactivity as an indicator or a methodwherein incorporation of bromodeoxiuridine (BrdU) i.e. a derivative ofthymidine is quantified by using an antibody specific to BrdU. In analternative method, production of various cytokines accompanying theactivation of immunocytes may be examined. Specifically, interleukins(IL-1, IL-2, IL-3I, L-4 etc.) and interferons (alpha, beta, gamma) TNF,GM-CSF, and various chemokines to be secreted into a medium or serumupon activation of immunocytes are measured by using antibodies specificthereto.

The protein of the invention includes e.g. proteins called muteincontaining (1) an amino acid sequence set forth in SEQ ID NO:4 wherein 1to 5 (preferably 1 to 3) amino acids are deleted, (2) an amino acidsequence set forth in SEQ ID NO:4 wherein 1 to 10 (preferably 1 to 5(more preferably 1 to 3)) amino acids are added, (3) an amino acidsequence set forth in SEQ ID NO:4 wherein 1 to 5 (preferably 1 to 3)amino acids are inserted, (4) an amino acid sequence set forth in SEQ IDNO:4 wherein 1 to 5 (preferably 1 to 3) amino acids are replaced byother amino acids, or (5) a combination of these amino acid sequences.

If the amino acid sequence has an insertion, deletion or substitution asdescribed above, the position of the insertion, deletion or substitutionis not particularly limited.

In this specification, the N-terminal (amino terminal) of the protein isplaced in the left and the C-terminal (carboxyl terminal) in the rightin accordance with a peptide notation system. The protein of theinvention, including the protein having the amino acid sequence setforth in SEQ ID NO:4, usually has a carboxyl group (—COOH) orcarboxylate (—COO—) at the C-terminal, but may have an amide (—CONH) orester (—COOR) at the C-terminal.

The group R in the ester includes e.g. C₁₋₆ alkyl groups such as methyl,ethyl, n-propyl, isopropyl and n-butyl, C₃₋₈ cycloalkyl groups such ascyclopentyl and cyclohexyl, C₆₋₁₂ aryl groups such as phenyl andα-naphthyl, and C₇₋₁₄ aralkyl groups, for example, phenyl-C₁₋₂ alkylgroups such as benzyl and phenetyl and α-naphthyl-C₁₋₂ alkyl groups suchas α-naphthylmethyl, as well as a pivaloyloxymethyl group used generallyfor oral administration.

When the protein of the invention has a carboxyl group (or carboxylate)at other position than the C-terminal, the protein of the inventionincludes those proteins wherein said carboxyl group is amidated oresterified. The ester used in this case includes e.g. the ester at theC-terminal as described above.

Also, the protein of the invention includes those proteins wherein anamino group of an N-terminal amino acid residue (e.g. methionineresidue) is protected with a protective group (e.g., C₁₋₆ acyl groupincluding C₁₋₆ alkanoyl such as formyl group and acetyl group), anN-terminal glutamic residue formed in vivo by cleavage is oxidized intopyroglutamine, or a substituent (e.g., —OH, —SH, amino group, imidazolegroup, indole group, guanidino group or the like) on a side chain of anintramolecular amino acid is protected with a suitable protective group(e.g., C₁₋₆ acyl group including C₁₋₆ alkanoyl such as formyl group andacetyl group), as well as conjugated proteins such as glycoproteinhaving sugar chains bound thereto.

As the protein of the invention, for example a human-derived (preferablyhuman kidney-derived) protein having the amino acid sequence set forthin SEQ ID NO:4 is used.

The partial peptide of the protein of the invention may be any partialpeptide which is derived from the protein of the invention describedabove and preferably has similar properties (for example, the activityof binding to NKG2D, the action of activating immunocytes etc.) to thoseof the protein of the invention described above. As the partial peptideof the invention, use is made of peptides having at least 20%,preferably 50% or more, more preferably 70% or more, still morepreferably 90% or more, and most preferably 95% or more of the aminoacid sequence constituting the protein of the invention and having theactivity of binding to NKG2D or the action of activating immunocytes.

Also, the partial peptide of the invention may have deletion of 1 to 5(preferably 1 to 3) amino acids in the amino acid sequence, addition of1 to 10 (preferably 1 to 5 (more preferably 1 to 3)) amino acids in theamino acid sequence, insertion of 1 to 5 (preferably 1 to 3) amino acidsin the amino acid sequence, or replacement of 1 to 5 (preferably 1 to 3)amino acids by other amino acids in the amino acid sequence.

Specifically, the partial peptide of the invention includes a partialpeptide having a partial amino acid sequence between positions 24 to 255in the amino acid sequence shown in SEQ ID NO:4, a partial peptidehaving a partial amino acid sequence between positions 31 to 255 in theamino acid sequence shown in SEQ ID NO:4, a partial peptide having apartial amino acid sequence between positions 26 to 255 in the aminoacid sequence shown in SEQ ID NO:4, a partial peptide having a partialamino acid sequence between positions 25 to 255 in the amino acidsequence shown in SEQ ID NO:4, and a partial peptide having a partialamino acid sequence between positions 27 to 255 in the amino acidsequence shown in SEQ ID NO:4.

The partial peptide of the invention usually has a carboxyl group(—COOH) or carboxylate (—COO—) at the C-terminal, but like the proteinof the invention described above, the partial peptide may have an amide(—CONH) or ester (—COOR) (R has the same meaning as defined above) atthe C-terminal.

Like the protein of the invention described above, the partial peptideof the invention includes those peptides wherein an amino group of anN-terminal amino acid residue (e.g. methionine residue) is protectedwith a protective group, an N-terminal glutamine residue is formed invivo by cleavage of the N-terminal and converted into pyroglutamic acid,or a substituent on a side chain of an intramolecular amino acid isprotected with a suitable protective group, as well as conjugatedpeptides such as glycopeptide having sugar chains bound thereto.

Because the partial peptide of the invention can be used as an antigenfor raising an antibody, it is not always necessary that the partialpeptide has the activity of binding to NKG2D or the action of activatingimmunocytes.

The protein of the invention, its partial peptide, or DNA coding for theprotein of the invention or its partial peptide may be labeled in amethod known per se, and includes e.g. those labeled with an isotope,with a fluorescent label (for example, a fluorescent label such asfluorescein), with biotin, with an enzyme etc.

As the salts of the protein or partial peptide of the invention, saltswith physiologically acceptable acids (e.g., inorganic acids, organicacids) or bases (e.g., alkali metal salts) are used, and in particularphysiologically acceptable acid-addition salts are preferable. Suchsalts include e.g. salts with inorganic acids (e.g., hydrochloric acid,phosphoric acid, hydrobromic acid, sulfuric acid) or salts with organicacids (e.g., acetic acid, formic acid, propionic acid, fumaric acid,maleic acid, succinic acid, tartaric acid, citric acid, malic acid,oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The protein or partial peptide of the invention or salts thereof can beproduced by known protein purification techniques from the human orwarm-blooded animal cells or tissues (particularly kidney etc.)described above, or by culturing transformants containing DNA coding forthe protein as described later. Alternatively, these can also beproduced by a peptide synthesis method described below.

For production from human or mammalian tissues or cells (particularlykidney etc.), the human or mammalian tissues or cells are homogenizedand then extracted with e.g. an acid, and the extract can be purifiedand isolated by a combination of chromatographic techniques such asreverse-phase chromatography, ion-exchange chromatography etc.

For production from human or mammalian tissues or cells (particularlykidney etc.), the human or mammalian tissues or cells are homogenizedand then extracted with e.g. an acid, and the extract can be purifiedand isolated by a combination of chromatographic techniques such asreverse-phase chromatography, ion-exchange chromatography etc.

For synthesis of the protein or partial peptide of the invention orsalts thereof or amide derivatives thereof, usually commerciallyavailable resin for protein synthesis can be used. Such resin includese.g. chloromethyl resin, hydroxymethyl resin, benzhydryl amine resin,aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethyl methyl phenyl acetamide methylresin, polyacrylamide resin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin, 4-(2′,4′-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resinetc. On the resin described above, each amino acid whose α-amino groupand side-chain functional group are properly protected is condensedsequentially in accordance with the sequence of the desired protein byvarious condensation methods known per se. At the end of the reaction,the protein is cleaved off from the resin while various protectivegroups are removed, and the product is subjected to an intramoleculardisulfide bond-forming reaction in a highly diluted solution to give thedesired protein or an amide thereof.

Although a wide variety of activating reagents usable for proteinsynthesis can be used for condensation of the protected amino acidsdescribed above, carbodiimides are particularly preferable. Examples ofsuch carbodiimides include DCC, N,N′-diisopropylcarbodiimide,N-ethyl-N′-(3-dimethylaminoprolyl) carbodiimide, etc. For activation bythese reagents, the protected amino acids along with racemizationinhibitors (e.g., HOBt, HOOBt) can be added to the resin directly orafter the protected amino acids are previously activated as symmetricacid anhydrides or HOBt esters or HOOBt esters.

The solvent used for activation of each protected amino acid or forcondensation thereof with the resin can be selected as necessary fromthose solvents known to be usable in protein condensation reaction.Examples of such solvent include acid amides such asN,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidone,hydrocarbon halides such as methylene chloride and chloroform, alcoholssuch as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, etherssuch as pyridine, dioxane and tetrahydrofuran, nitriles such asacetonitrile and propionitrile, esters such as methyl acetate and ethylacetate, or a suitable mixture thereof. The reaction temperature isusually selected as necessary within the range known to be usable in thereaction of forming protein bonds, and usually the reaction temperatureis selected within the range of −20° C. to 50° C. The activated aminoacid derivatives are used usually in excess (1.5- to 4-fold). When thecondensation is insufficient as a result of a test using ninhydrinreaction, the condensation reaction is repeatedly carried out withoutconducting elimination of the protective groups, whereby sufficientcondensation can be achieved. When sufficient condensation cannot beachieved even by repeatedly carrying out the reaction, the unreactedamino acids are acetylated with acetic anhydride or acetyl imidazole sothat the subsequent reaction cannot be influenced.

The protective groups for amino groups in the raw materials include e.g.Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl—Z, Br—Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl,diphenylphospinothioyl, Fmoc etc.

The carboxyl group can be protected by e.g. alkyl esterification (e.g.,linear, branched or cyclic alkyl esterification such as methyl, ethyl,propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloctylor 2-adamantyl esterification), aralkyl esterification (e.g., benzylesterification, 4-nitrobenzyl esterification, 4-methoxybenzylesterification, 4-chlorobenzyl esterification, benzhydrylesterification), phenacyl esterification, benzyloxy carbonylhydrazidation, t-butoxycarbonyl hydrazidation, trityl hydrazidation etc.

The hydroxyl group in serine can be protected by e.g. esterification oretherification. A suitable group used in this esterification includese.g. lower (C₁₋₆) alkanoyl groups such as acetyl group, aroyl groupssuch as benzoyl group, and carbonic acid-derived groups such asbenzyloxycarbonyl group and ethoxycarbonyl group. A suitable group foretherification includes e.g. a benzyl group, tetrahydropyranyl group,t-butyl group etc.

The protective group used for the phenolic hydroxyl group in tyrosineincludes e.g. Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br—Z, t-butyl etc.

The protective group used for imidazole in histidine includes e.g. Tos,4-methoxy-2,3,6-trimethylbenzene sulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, Fmoc etc.

The raw materials having activated carboxyl groups include e.g. thecorresponding acid anhydrides, azides and active esters (i.e. esterswith alcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide and HOBt). The raw materialshaving activated amino groups include e.g. the corresponding phosphoricacid amides.

The method for removal (elimination) of the protective groups makes useof e.g. catalytic reduction in a hydrogen stream in the presence of acatalyst such as Pd-black or Pd-carbon, acid treatment with anhydroushydrogen fluoride, methane sulfonic acid, trifluoromethane sulfonicacid, trifluoroacetic acid or a mixed solution thereof, base treatmentwith diisopropylethylamine, triethylamine, piperidine or piperazine, andreduction with sodium in liquid ammonia. The elimination reaction by theacid treatment is carried out generally at a temperature of about −20°C. to 40° C., and in the acid treatment, addition of cation scavengerssuch as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butane dithiol and 1,2-ethane dithiol is effective. A2,4-dinitrophenyl group used as a protective group for imidazole inhistidine can also be removed by treatment with thiophenol, while aformyl group used as a protective group for indole in tryptophan can beremoved not only by deprotection by acid treatment in the presence of1,2-ethane dithiol or 1,4-butane dithiol, but also by alkali treatmentwith a diluted sodium hydroxide solution or diluted ammonia.

Protection and protective groups for functional groups which should notparticipate in the reaction of the raw materials, elimination of theprotective groups, and activation of functional groups participating inthe reaction can be selected as necessary from known groups or knownmeans.

Another method of obtaining an amide derivative of the protein involvesprotecting the a-carboxyl group of a C-terminal amino acid by amidation,then extending a peptide (protein) chain at the side of the amino groupuntil it attains desired chain length, and thereafter producing aprotein of said peptide chain from which only the protective group forthe N-terminal α-amino group has been removed and a protein of saidpeptide chain from which only the protective group for the C-terminalcarboxyl group has been removed, followed by condensation of both theproteins in the mixed solvent described above. The details of thecondensation reaction are the same as described above. The protectedprotein obtained by condensation is purified, and every protective groupis removed by the method descried above, whereby the desired crudeprotein can be obtained. This crude protein is purified by a widevariety of known purification techniques, and by lyophilizing its mainfraction, the desired amide derivative of the protein can be obtained.

To obtain an ester derivative of the protein, for example the α-carboxylgroup of a C-terminal amino acid is condensed with desired alcohol toform an amino acid ester from which the desired ester derivative of theprotein can be obtained in the same manner as for the amide derivativeof the protein.

The partial peptide of the invention or salts thereof can be producedaccording to a peptide synthesis method known per se or by cleaving theprotein of the invention with a suitable peptidase. For example, thepeptide synthesis method may be the solid- or liquid-phase synthesismethod. That is, the desired peptide can be obtained by condensation ofa partial peptide or amino acids capable of constituting the partialpeptide of the invention with the remainder, followed by elimination ofprotective groups if any from the product. As the known condensationmethod and the elimination of the protective groups, mention is made ofe.g. the methods described in (1) to (5) below:

-   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York (1966);-   (2) Schroeder and Luebke, The Peptide, Academic Press, New York    (1965);-   (3) N. Izumiya et al., Basis and Experiments in Peptide Synthesis    (in Japanese), Maruzen K. K. (1975);-   (4) T. Yajima & S. Sakakibara, Biochemical Experimental Lecture 1,    Protein Chemistry IV, 205 (1977) (in Japanese); and-   (5) A Sequel to Development of Pharmaceutical Preparations, Vol. 14,    Peptide Synthesis (in Japanese), supervised by T. Yajima, Hirokawa    Shoten.

Further, after the reaction, the partial peptide of the invention can beisolated and purified by a combination of conventional purificationtechniques such as solvent extraction, distillation, columnchromatography, liquid chromatography and recrystallization. If thepartial peptide is obtained in a free form by these methods, the productcan be converted into a suitable salt by a known method or its analogousmethod, or if the partial peptide is obtained in a salt form, it can beconverted into a free peptide or other salts by a known method or itsanalogous method.

The DNA coding for the protein of the invention may be any DNAcontaining a nucleotide sequence coding for the protein of the inventiondescribed above. This DNA may also be genomic DNA, a genomic DNAlibrary, cDNA derived form the above-described cells or tissues, a cDNAlibrary derived form the above-described cells or tissues, or syntheticDNA.

The vector used in the library may be bacteriophage, plasmid, cosmid,phagimide or the like. A total RNA or mRNA fraction prepared from thecells and tissues described above can also be used in directamplification by Reverse Transcriptase Polymerase Chain Reaction(abbreviated hereinafter to RT-PCR method).

The DNA coding for the protein of the invention includes e.g. DNAcontaining DNA having the nucleotide sequence set forth in SEQ ID NO:3,or DNA having a nucleotide sequence hybridizing under high stringentconditions with the nucleotide sequence set forth in SEQ ID NO:3 andcoding for a protein having substantially identical properties (e.g.,the activity of binding to NKG2D, the action of activating immunocytes,etc.) with those of the protein of the invention.

The high stringent conditions refer to those conditions under which theconcentration of sodium is about 19 to 40 mM, preferably about 19 to 20mM, and the temperature is about 50 to 70° C., preferably about 60 to65° C. In particular, the high stringent conditions are preferably thoseconditions under which the concentration of sodium is about 19 mM, andthe temperature is about 65° C.

Specifically, the DNA coding for a protein having the amino acidsequence set forth in SEQ ID NO:4 includes DNA having the nucleotidesequence set forth in SEQ ID NO:3.

The DNA coding for the partial peptide of the invention may be any DNAcontaining a nucleotide sequence encoding the above-described partialpeptide of the invention. It may also be genomic DNA, a genomic DNAlibrary, cDNA derived form the above-described cells or tissues, a cDNAlibrary derived form the above-described cells or tissues, or syntheticDNA.

The DNA coding for the partial peptide of the invention includes e.g.DNA having a partial nucleotide sequence of DNA having the nucleotidesequence set forth in SEQ ID NO:3, or DNA having a nucleotide sequencehybridizing under high stringent conditions with the nucleotide sequenceset forth in SEQ ID NO:3 and having a partial nucleotide sequence of DNAcoding for a protein having substantially identical properties withthose of the protein of the invention.

As a means of cloning the DNA coding completely for the protein andpartial peptide of the invention (these proteins are referred tocollectively as the protein of the invention in the followingdescription of cloning and expression of the DNA coding for theseproteins), it is possible to use amplification by the known PCR methodusing synthetic DNA primers having a partial nucleotide sequence of theprotein of the invention, or selection by hybridization of the DNAintegrated in a suitable vector with a labeled DNA fragment or syntheticDNA coding for a part or the whole of the protein of the invention.Hybridization can be carried out according to a method described in e.g.Molecular Cloning, 2nd ed., J. Sambrook et al., Cold Spring Harbor Lab.Press, 1989. When a commercial library is used, hybridization can becarried out according to its accompanying protocol.

Conversion of the nucleotide sequence of DNA can be carried out by amethod known per se, for example by the Gupped duplex method or Kunkelmethod or its analogous method by using a known kit such as Mutan™-G(Takara Shuzo K. K.) or Mutan T-K (Takara Shuzo K. K.).

The cloned DNA coding for the protein may be used as such depending onthe object or if necessary after digestion with restriction enzymes orafter addition of a linker. The DNA may have ATG as a translationinitiation codon at the 5′-terminal thereof and TAA, TGA or TAG as atranslation termination codon at the 3′-terminal thereof Thesetranslation initiation and termination codons can also be added to theDNA via a suitable synthetic DNA adaptor.

An expression vector for the protein of the invention can be producedfor example by (A) cutting a desired DNA fragment off from the DNAcoding for the protein of the invention and then (B) ligating the DNAfragment to a region downstream from a promoter in a suitable expressionvector.

The vector used includes E. coli-derived plasmids (e.g., pBR322, pBR325,pUC12, pUC13), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5,pC194), yeast-derived plasmids (e.g., pSH19, pSH15), bacteriophage suchas λ-phage, and animal viruses such as retrovirus, vaccinia virus andbaculovirus, as well as pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo etc.

The promoter used in this invention may be any suitable promotercompatible with a host used for expression of the gene. For example,when animal cells are used as the host, mention is made of SRα promoter,SV40 early promoter, HIV-LTR promoter, CMV promoter, HSV-TK promoteretc.

Among these promoters, CMV (cytomegalovirus) promoter, SRα promoter etc.are preferably used. It is preferable to use trp promoter, lac promoter,recA promoter, λPL promoter, lpp promoter, T7 promoter etc. formicroorganisms of the genus Escherichia as the host, SPO1 promoter, SPO2promoter, penP promoter etc. for microorganisms of the genus Bacillus asthe host, and PHO5 promoter, PGK promoter, GAP promoter, ADH promoteretc. for yeasts as the host. When insect cells are used as the host,polyhedron promoter, P10 promoter etc. are preferable.

The expression vector may contain an enhancer, a splicing signal, a polyA-added signal, a selective marker, an SV40 origin of replication (alsoreferred to hereinafter as SV40 ori) etc. if necessary in addition tothe element described above. The selective marker includes e.g.dihydrofolate reductase (also referred to hereinafter as dhfr) gene[methotrexate (MTX) resistance], ampicillin resistance gene (alsoreferred to hereinafter as Amp^(r)) and neomycin resistance gene (G418resistance, also referred to hereinafter as Neo^(r)). In particular,when the dhfr gene is used as a selective marker for dhfr gene-defectiveChinese hamster cells, the desired gene can also be selected in athymidine-free medium.

A signal sequence compatible with the host is added if necessary to thesequence corresponding to the N-terminal of the protein of theinvention. A PhoA signal sequence, Omp A signal sequence etc. can beutilized for microorganisms of the genus Escherichia used as the host;an α-amylase signal sequence, subtilisin signal sequence etc. formicroorganisms of the genus Bacillus as the host; an MFα signalsequence, SUC2 signal sequence etc. for yeasts as the host; and aninsulin signal sequence, α-interferon signal sequence, antibody moleculesignal sequence etc. for animal cells as the host.

The thus constructed vector containing the DNA coding for the protein ofthe invention can be used to produce transformants.

Microorganisms of the genus Escherichia, microorganisms of the genusBacillus, yeasts, insect cells, insects, animal cells etc. are used asthe host.

The microorganisms of the genus Escherichia used include e.g.Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci. USA, vol. 60, 160(1968)), JM103 (Nucleic Acids Research, vol. 9, 309 (1981)), JA221(Journal of Molecular Biology, vol. 120, 517 (1978)), HB101 (Journal ofMolecular Biology, vol. 41, 459 (1969)), C600 (Genetics, vol. 39, 440(1954)), etc.

The microorganisms of the genus Bacillus used include e.g. Bacillussubtilis MI114 (Gene, vol. 24, 255 (1983)), 207-21 (Journal ofBiochemistry, vol. 95, 87 (1984)), etc.

The yeasts used include Saccharomyces cerevisiae AH22, AH22R⁻, NA87-l1A,DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichiapastoris KM71, etc.

For example, when the virus is AcNPV, the insect cells used include e.g.cells (Spodoptera frugiperda cells; Sf cells) from an established cellline derived from caterpillars of Spodoptera frugiperda, MG1 cellsderived from the midgut in Trichoplusia ni, High Five™ cells derivedfrom eggs of Trichoplusia ni, cells derived from Mamestra brassicae andcells derived from Estigmena acrea. When the virus is BmNPV, cells(Bombyx mori N cells; BmN cells) from an established cell line derivedfrom silkworms, etc., are used. The Sf cells used include e.g. Sf9 cells(ATCC CRL1711), Sf21 cells (Vaughn, J. L. et al., In Vivo, 13, 213-217(1977)), etc.

The insects used include e.g. silkworm caterpillars (Maeda et al.,Nature, vol. 315, 592 (1985)).

The animals cells used include e.g. simian cell COS-7 (COS-7), Vero,Chinese hamster ovary cells CHO (abbreviated hereinafter to CHO cells),dhfr gene-defective Chinese hamster cells CHO (abbreviated hereinafterto CHO (dhfr⁻) cells), mouse L cells, mouse AtT-20, mouse myeloma cells,rat GH3, human FL cells etc. Further, use can also be made of variousnormal human cells such as hepatic cells, spleen cells, nerve cells,glia cells, pancreatic β-cells, bone marrow cells, mesangial cells,Langerhans cells, epidermal cells, epithelial cells, endothelial cells,fibroblasts, fibrous cells, muscular cells, adipocytes, immunocytes(e.g., macrophage, T cells, B cells, natural killer cells, mast cells,neutrophils, basophils, acidophils, monocytes), megakaryocytes, synovialmembrane cells, cartilage cells, bone cells, osteoblasts, osteoclasts,mammary gland cells, hepatic cells or interstitial cells, or theirprecursor cells, stem cells or cancer cells).

The microorganisms of the genus Escherichia can be transformed accordingto a method described in e.g. Proc. Natl. Acad. Sci. USA, vol. 69, 2110(1972) or Gene, vol. 17, 107 (1982).

The microorganisms of the genus Bacillus can be transformed according toa method described in e.g. Molecular & General Genetics, vol. 168, 111(1979), etc.

The yeasts can be transformed according to a method described in e.g.Methods in Enzymology, vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci.USA, vol. 75, 1929 (1978), etc.

The insect cells or insects can be transformed according to a methoddescribed in e.g. Bio/Technology, 6, 47-55 (1988), etc.

The animal cells can be transformed according to a method described ine.g. Cell Technology, Extra Number 8, New Experimental Protocol in CellTechnology”, 263-267 (1995) (published by Shujunsha), Virology, vol. 52,456 (1973), etc.

Transformants transformed with the expression vector containing DNAcoding for the protein can be thus obtained.

When transformants derived from the microorganisms of the genusEscherichia or Bacillus as the host are cultured, the medium used fortheir culture is preferably a liquid medium containing a carbon source,a nitrogen source, inorganic matter etc. necessary for growth of thetransformants. The carbon source includes e.g. glucose, dextrin, solublestarch, sucrose etc.; the nitrogen source includes e.g. inorganic ororganic materials such as ammonium salts, nitrates, corn steep liquor,peptone, casein, meat extract, soybean cake and potato extract; and theinorganic matter includes e.g. calcium chloride, sodium dihydrogenphosphate, magnesium chloride etc. In addition, a yeast extract,vitamins, growth promoting factors etc. may be added. The pH value ofthe medium is desirably about 5 to 8.

For example, the medium for culturing the microorganisms of the genusEscherichia is preferably M9 medium containing glucose and casamino acid(Miller, Journal of Experiments in Molecular Genetics, 431-433, ColdSpring Harbor Laboratory, New York, 1972). To permit the promoter towork efficiently if necessary, a chemical such as 3β-indolylacrylic acidcan be added thereto.

The transformants from the microorganisms of the genus Escherichia asthe host are cultured usually at about 15 to 43° C. for about 3 to 24hours during which the medium may be aerated or stirred as necessary.

The transformants from the microorganisms of the genus Bacillus as thehost are cultured usually at about 30 to 40° C. for about 6 to 24 hoursduring which the medium may be aerated or stirred as necessary.

The medium used for culturing the transformants from yeasts as the hostincludes e.g. Burkholder minimum medium (Bostian, K. L. et al., Proc.Natl. Acad. Sci. USA, vol. 77, 4505 (1980)) and SD medium containing0.5% casamino acid (Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA,vol. 81, 5330 (1984)). The pH value of the medium is adjusted preferablyto about 5-8. The transformants are cultured usually at about 20 to 35°C. for about 24 to 72 hours during which the medium may be aerated orstirred as necessary.

The medium used for culturing the transformants from insect cells orinsects as the host includes e. g. a medium prepared by addinginactivated additives such as 10% bovine serum to Grace's insect medium(Grace, T. C. C., Nature, 195, 788 (1962)). The pH value of the mediumis adjusted preferably to about 6.2-6.4. The transformants are culturedusually at about 27° C. for about 3 to 5 days during which the mediummay be aerated or stirred as necessary.

The medium used for culturing the transformants from animal cells as thehost includes e.g. MEM medium containing about 5 to 20% FBS (Science,122, 501 (1952)), DMEM medium (Virology, vol. 8, 396 (1959)), RPMI 1640medium (The Journal of the American Medical Association, vol. 199, 519(1967)), 199 medium (Proceeding of the Society for the BiologicalMedicine, vol. 73, 1 (1950)) etc. The pH value is preferably about 6 to8. The transformants are cultured usually at about 30 to 40° C. forabout 15 to 60 hours during which the medium may be aerated or stirredas necessary.

Thus, the protein of the invention can be formed in the cells or out ofthe cells of the transformants.

From the resulting culture, the protein of the invention can beseparated and purified for example in the following manner.

To extract the protein of the invention from the cultured microorganismsor cells, the cultured microorganisms or cells are collected in a usualmanner, suspended in a suitable buffer, disrupted by sonication,lysozyme and/or freezing and thawing, and centrifuged or filtered togive a crude extract of the protein. The buffer may contain proteindenaturants such as urea and guanidine hydrochloride and surfactantssuch as Triton X-100™. When the protein is secreted into the cultureliquid, the culture supernatant is collected by separating thesupernatant from the cultured microorganisms or cells by a method knownper se.

The culture supernatant thus obtained, or the protein contained in theextract, can be purified by a suitable combination of separation andpurification techniques known per se. These known separation andpurification techniques make use of a method of utilizing solubility,such as salting-out and solvent precipitation, a method of mainlyutilizing a difference in molecular weight, such as dialysis,ultrafiltration, gel filtration, and SDS-polyacrylamide gelelectrophoresis, a method of utilizing a difference in electric charge,such as ion-exchange chromatography, a method of utilizing specificaffinity, such as affinity chromatography, a method of utilizing adifference in hydrophobicity, such as reverse-phase HPLC, a method ofutilizing a difference in isoelectric point, such as isoelectricfocusing.

If the protein thus obtained is in a free form, it can be converted intoa salt by a method known per se or its analogous method, while if theresulting protein is obtained in the form of a salt, it can be convertedinto a free protein or another salt by a method known per se or itsanalogous method.

Before or after purification, a suitable protein-modifying enzyme may beact on act on the protein produced by the transformants, whereby theprotein can be arbitrarily modified or a partial polypeptide can beremoved therefrom. For example, trypsin, chymotrypsin, arginylendopeptidase, protein kinase or glycosidase is used as theprotein-modifying enzyme.

The presence or activity of the thus formed protein of the invention orsalts thereof can be measured by an experiment of binding with a labeledligand and enzyme immunoassays with specific antibody.

The antibody against the protein and partial peptide of the invention orsalts thereof may be a polyclonal or monoclonal antibody capable ofrecognizing the protein or partial peptide of the invention or saltsthereof The antibody against the protein and partial peptide of theinvention or salts thereof (referred to collectively as the protein ofthe invention in the following description of the antibody) can beproduced by a known process for producing antibody or antiserum by usingthe protein of the invention as the antigen.

[Preparation of the Monoclonal Antibody]

(a) Preparation of Monoclonal Antibody-Producing Cells

The protein of the invention is administered alone or together with acarrier and a diluent into warm-blooded animals at a site where theantibody can be produced by administration. To enhance the ability ofthe animals upon administration to produce the antibody, completeFreund's adjuvant or incomplete Freund's adjuvant may be administered.Administration is conducted usually once every 2 to 6 weeks and about 2to 10 times in total. The warm-blooded animals used include e.g. monkey,rabbit, dog, guinea pig, mouse, rat, sheep, goat and chicken, amongwhich mouse and rat are preferably used.

For production of the monoclonal antibody-producing cells, those animalshaving antibody titer are selected from the warm-blooded animals (e.g.mice) immunized with the antigen, and on the second to fifth day afterthe final immunization, their spleens or lymph nodes are collected, andthe antibody-producing cells contained therein are fused with myelomacells from animals of the same or different species, whereby monoclonalantibody-producing hybridomas can be produced. The antibody titer inantiserum can be measured for example by reacting the antiserum with alabeled protein described later and then measuring the activity of alabel bound to the antibody. Fusion can be carried out by a known methodsuch as the method of Kohler and Millstein (Nature, 256, 495 (1975)).The fusion promoter includes polyethylene glycol (PEG) and Sendai virus,and PEG is preferably used.

The myeloma cells include myeloma cells NS-1, P3U1, SP2/0 and AP-1 fromwarm-blooded animals, among which P3U1 is preferably used. The ratio ofthe antibody-producing cells (spleen cells) to the myeloma cells used isfrom about 1:1 to 20:1, and cell fusion can be effected efficiently byincubating the cells for about 1 to 10 minutes at 20 to 40° C.,preferably 30 to 37° C., in the presence of PEG (preferably PEG 1000 toPEG 6000) at a concentration of about 10 to 80%.

The monoclonal antibody-producing hybridoma can be screened by variousmethods, for example by adding a culture supernatant of the hybridoma toa solid phase (e.g., a microplate) having the protein antibody adsorbedthereon directly or along with a carrier and then adding a radioactivesubstance- or enzyme-labeled anti-immunoglobulin antibody (which is e.g.an anti-mouse immunoglobulin antibody when mouse cells are subjected tocell fusion) or protein A to detect the monoclonal antibody bound to thesolid phase or by adding a culture supernatant of the hybridoma to asolid phase having an anti-immunoglobulin antibody or protein A adsorbedthereon and then adding the protein labeled with a radioactive substanceor an enzyme to detect the monoclonal antibody bound to the solid phase.

The monoclonal antibody can be screened in a method known per se or itsanalogous method. Screening can be carried out usually in an animal cellculture medium to which HAT (hypoxanthine, aminopterin, thymidine) wasadded. The screening and breeding medium may be any medium in which thehybridoma can grow. Examples of such medium include PRMI 1640 mediumcontaining 1 to 20% (preferably 10 to 20%) FBS, GIT medium containing 1to 10% FBS (Wako Pure Chemical Industries, Ltd.), and a serum-freemedium for hybridoma culture (SFM-101, Nissui Seiyaku K. K.). Theculture temperature is usually 20 to 40° C., preferably about 37° C. Theculture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks.Culture can be conducted usually in 5% CO₂ gas. The antibody titer in aculture supernatant of the hybridoma can be measured in the same manneras in the measurement of the antibody titer in antiserum as describedabove.

(b) Purification of the Monoclonal Antibody

The monoclonal antibody can be separated and purified by techniquesknown per se, for example by techniques of separating and purifyingimmunoglobulins, such as salting-out, alcohol precipitation, isoelectricprecipitation, electrophoresis, absorption-desorption with anion-exchanger (e.g. DEAE), ultracentrifugation and gel filtration or byspecific purification of the antibody by collecting it with an activeadsorbent such as antigen-bound solid phase, protein A or protein G andthen eluting the antibody therefrom.

[Preparation of the Polyclonal Antibody]

The polyclonal antibody of the invention can be produced by a methodknown per se or its analogous method. For example, the desiredpolyclonal antibody can be produced by preparing the immune antigen(protein antigen) or a conjugate thereof with a carrier protein, thenimmunizing warm-blooded animals therewith in the same manner as inproduction of the monoclonal antibody as described above, collecting amaterial containing the antibody against the protein of the inventionfrom the immunized animals, and separating and purifying the antibody.

For the conjugate of the immune antigen with a carrier protein used forimmunizing warm-blooded animals, the type of the carrier protein and themixing ratio of the carrier to the hapten are not particularly limitedinsofar as the desired antibody can be efficiently produced byimmunization with the hapten crosslinked with the carrier, and forexample, the conjugate is produced by coupling the hapten with bovineserum albumin, bovine cyloglobulin, hemocyanin or the like in a ratio ofabout 0.1-20:1, preferably about 1-5:1.

The hapten can be coupled with the carrier by use of variouscondensation agents such as glutaraldehyde, carbodiimide,maleimide-activated ester, and activated ester reagents containing thiolgroup and dithiopyridyl group.

The resulting condensation product is administered alone or togetherwith a carrier and a diluent into warm-blooded animals at a site wherethe antibody can be produced. To enhance the ability of the animals uponadministration to produce the antibody, complete Freund's adjuvant orincomplete Freund's adjuvant may be administered. Administration isconducted usually once every 2 to 6 weeks and about 3 to 10 times intotal.

The polyclonal antibody can be collected form blood, ascites etc.preferably blood in the warm-blooded animals immunized by the methoddescribed above.

The polyclonal antibody titer in antiserum can be measured in the samemanner as in the measurement of the antibody titer in antiserum asdescribed above. Separation and purification of the polyclonal antibodycan be carried out by a method of separating and purifyingimmunoglobulins, which is similar to the separation and purification ofthe monoclonal antibody as described above.

Hereinafter, use of the protein or partial peptide of the invention orsalts thereof (also referred to hereinafter as the protein etc. of theinvention) and the antibody against the protein or partial peptide ofthe invention or against salts thereof (also referred to hereinafter asthe antibody of the invention) is described.

(1) An Agent for Treating and Preventing Various Diseases, WhichContains the Protein etc. of the Invention

The protein etc. of the invention have the activity of binding to NKG2Dwhich is a receptor observed to be expressed in immunocytes etc. NKG2Dis expressed in mainly NK cells and also in a part of T cells, and knownas a receptor activating these cells. Accordingly, the protein etc. ofthe invention can be expected to activate immunocytes via NKG2D. Theprotein etc. of the invention can be used in therapy and prevention ofvarious diseases thorough its immune activating activity. That is, theprotein etc. of the invention can be used not only in an agent fortreating and preventing diseases caused by infection withmicroorganisms, new microorganisms and viruses but also in an agent fortreating and preventing diseases such as various cancers (for example,uterine cancer, endometrium tumor, breast cancer, colon cancer, prostatecancer, lung cancer, kidney cancer, neuroblastoma, bladder cancer,melanoma etc.). In particular, the protein etc. of the invention isuseful as a medicine for prevention of recurrence and complete treatmentof various cancers after excision of carcinoma by its immune activatingaction.

When the protein etc. of the invention are used as the therapeutic orpreventive agent described above, the protein etc. of the invention arepurified to a purity of at least 90%, preferably 95% or more, morepreferably 98% or more, still more preferably 99% or more.

For example, the protein etc. of the invention can be used orally astablets coated with sugar as necessary, capsules, elixirs andmicrocapsules or parenterally as an aseptic solution with water or withother pharmaceutically acceptable solutions or as an injection such assuspension. For example, these compositions can be produced by admixingphysiologically acceptable carriers, flavorings, excipients, vehicles,preservatives, stabilizers and binders with the protein etc. of theinvention in a unit dose required for generally approved pharmaceuticalmanufacturing. The amount of the active ingredient in thesepharmaceutical compositions is designed to have a suitable capacity inthe designated range.

The additives which can be admixed with the tablets, capsules etc.include e.g. binders such as gelatin, corn starch, tragacanth, Arabiagum, excipients such as crystalline cellulose, swelling agents such ascorn starch, gelatin and alginic acid, lubricants such as magnesiumstearate, sweeteners such as sucrose, lactose and saccharine, andflavorings such as peppermint, Gaultheria adenothrix oil and cherry.When one capsule is in a unit form, liquid carriers such as fats andoils can be contained in the materials described above. The asepticcomposition for injection can be formulated according to conventionalpharmaceutical manufacturing by dissolving or suspending the activematerial and naturally occurring vegetable oils such as sesame oil andcoconut oil in vehicles such as injection water.

The aqueous solution for injection includes e.g. physiological saline oran isotonic solution containing glucose and other auxiliary chemicals(e.g., D-sorbitol, D-mannitol, sodium chloride etc.), and may be used incombination with suitable solubilizers such as alcohols (e.g., ethanoletc.), polyalcohols (e.g., propylene glycol, polyethylene glycol etc.)and nonionic surfactants (e.g., Polysorbate 80™, HCO-50 etc.). The oilysolution includes e.g. sesame oil, soybean oil etc., and may be used incombination with solubilizers such as benzyl benzoate, benzyl alcoholetc. Further, it may be compounded with the buffer (e.g., phosphatebuffer, sodium acetate buffer etc.), analgesic agents (e.g.,benzalkonium chloride, procaine hydrochloride etc.), stabilizers (e.g.,human serum albumin, polyethylene glycol etc.), preservatives (e.g.,benzyl alcohol, phenol etc.), antioxidants etc. Usually, the preparedinjection is introduced into suitable ampoules.

The pharmaceutical composition thus obtained is safe and low toxic sothat it can be administered into e.g. humans or warm-blooded animals(e.g., rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse,cat, dog, monkey etc.).

The dose of the protein etc. of the invention is varied depending on theintended disease, the subject of administration etc., and when theprotein etc. of the invention are administered for example as ananticancer drug, the protein etc. are administered usually in a dose ofabout 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferablyabout 1.0 to 20 mg every 10 day into a (60 kg) adult.

(2) Screening of Candidates for Pharmaceutical Compounds AgainstDiseases

The compound or a salt thereof inhibiting the activity of the protein ofthe invention, that is, an antagonist for the protein of the invention,can be expected to suppress immunocytes. This antagonist can be used asan immunosupressant or an antiinflammatory agent not only againstvarious diseases such as autoimmune diseases and infectioushyper-immunoreactions, but also for suppression of immunoreactions aftertransplantation of organs and tissues.

Like the protein of the invention, the compound or a salt thereofpromoting the activity of the protein of the invention, that is, anagonist for the protein of the invention, can be used for treating andpreventing various diseases by its immune activating action. That is, itcan be used in pharmaceutical compositions including not only an agentfor treating and preventing diseases caused by infection withmicroorganisms, new microorganisms and viruses but also an agent fortreating and preventing diseases such as various cancers (for example,uterine cancer, endometrium tumor, breast cancer, colon cancer, prostatecancer, lung cancer, kidney cancer, neuroblastoma, bladder cancer,melanoma etc.).

Accordingly, the protein etc. of the invention are useful as a reagentfor screening a compound or a salt thereof having the activity ofpromoting or inhibiting the binding of the protein etc. of the inventionto NKG2D or the activation of immunocytes.

That is, this invention provides:

1. [1] a method for screening a compound having the activity ofpromoting or inhibiting the binding of the protein of the invention, apartial peptide thereof or a salt thereof to NKG2D or the activation ofimmunocytes (also referred to as a promoter or inhibitor in “(2)Screening for pharmaceutical candidates against diseases”),characterized in that the protein of the invention, a partial peptidethereof or a salt thereof is used (also referred to as the screeningmethod of this invention in “(2) Screening for pharmaceutical candidatesagainst diseases”) and [2] a kit for screening an inhibitor,characterized in the protein of the invention, a partial peptide or asalt thereof is contained (also referred to as the screening kit of theinvention in “(2) Screening for pharmaceutical candidates againstdiseases”), and more specifically

2. [1] a method of screening an inhibitor, characterized by comparingthe case (i) where a substrate is contacted with the protein of theinvention, a partial peptide thereof or a salt thereof with the case(ii) where the substrate and a test compound are contacted with theprotein of the invention, a partial peptide thereof or a salt thereof,and [2] a kit for screening an inhibitor, characterized in that theprotein of the invention, a partial peptide thereof or a salt thereofand a substrate are contained.

Specifically, the screening method and the screening kit described aboveare characterized in that for example, the binding of the protein etc.of the invention to NKG2D or the activation of immunocytes is measuredand compared between the cases (i) and (ii).

The binding of the protein etc. of the invention to NKG2D and theactivation of immunocytes can be measured in a method known per se orits analogous method.

The test compound includes e.g. peptides, proteins, non-peptidecompounds, synthetic compounds, fermentation products, cellularextracts, plant extracts, animal tissue extracts etc., and thesecompounds may be novel or known compounds.

The substrate may be any one which can serve as the substrate for theprotein etc. of the invention.

For example, a test compound showing higher inhibition of the binding toNKG2D or the activation of immunocytes in the case (ii) by about 20% ormore, preferably about 30% or more, more preferably about 50% or morethan in the case (i) can be selected as a compound inhibiting thebinding of the protein etc. of the invention to NKG2D or the activationof immunocytes.

Further, a test compound showing higher promotion of the binding toNKG2D or the activation of immunocytes in the case (ii) by about 20% ormore, preferably about 30% or more, more preferably about 50% or morethan in the case (i) can be selected as a compound promoting the bindingof the protein etc. of the invention to NKG2D or the activation ofimmunocytes.

The screening kit of the invention comprises the protein of theinvention, a partial peptide thereof or a salt thereof. For example, thescreening kit of the invention is as follows:

[Screening Reagents]

1. Measurement Buffers

Phosphate buffer containing FBS (fetal bovine serum)

Tris-HCl buffer, pH 7.5 (containing MgCl₂, EDTA)

2. Protein Standard

A fusion protein between the protein of the invention, a partial peptidethereof or a salt thereof and Fc

3. KG2D Source

KG2D-expressing CHO-K1 cells

4. Labeling Antibody

Anti-human IgG (Fc)-FITC conjugate

5. Detection

Flow cytometer

[Measurement Method]

The protein standard and a test compound are added to theKG2D-expressing CHO-K₁ cells, and then labeled with the anti-human IgG(Fc)-FITC conjugate, and the binding thereof to KG2D is measured with aflow cytometer.

The compounds or salts thereof obtained by the screening method orscreening kit of the invention are compounds selected from the abovetest compounds e.g. peptides, proteins, non-peptide compounds, syntheticcompounds, fermentation products, cellular extracts, plant extracts,animal tissue extracts, plasma etc., and these are compounds having theactivity of inhibiting the binding of the protein etc. of the inventionto NKG2D.

As the salts of said compounds, the same salts as those of the proteinof the invention described above are used.

When the compound obtained by the screening method or screening kit ofthe invention is used as the therapeutic or preventive agent describedabove, the compound can be used according to a conventional means. Forexample, the compound can be formed into tablets, capsules, elixirs,microcapsules, aseptic solutions and suspensions in the same manner asin production of the pharmaceutical composition containing the proteinetc. of the invention described above.

The pharmaceutical composition thus obtained is safe and low toxic sothat it can be administered into e.g. humans or warm-blooded animals(e.g., mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog,monkey etc.).

The dose of the compound or a salt thereof is varied depending on itsaction, the intended disease, the subject of administration,administration route etc., and when the compound having the activity ofinhibiting the cell-proliferating activity of the protein etc. of theinvention is orally administered for example for the purpose oftreatment of autoimmune diseases, said compound is administered in adose of usually about 0.1 to 100 mg, preferably about 1.0 to 50 mg, morepreferably about 1.0 to 20 mg every day into a (60 kg) adult. In thecase of parenteral administration, the dose of said compound is varieddepending on the subject of administration, the intended disease etc.,and when the compound having the activity of inhibiting thecell-proliferating activity of the protein etc. of the invention isadministered in the form of an injection for example for the purpose oftreatment of autoimmune diseases, said compound is conveniently injectedintravenously in a dose of usually about 0.01 to 30 mg, preferably about0.1 to 20 mg, more preferably about 0.1 to 10 mg every day into a (60kg) adult. Said compound can be administered into other animals in thesame dose per 60 kg as described above.

To carry out the screening method, the protein etc. of the invention issuspended in a buffer suitable for screening, whereby a preparation ofthe protein etc. of the invention is prepared. The buffer may be anybuffers such as phosphate buffer, Tris-HCl buffer etc. at a pH value ofabout 4 to 10 (desirably a pH value of about 6 to 8) which do notinhibit the reaction of the protein etc. of the invention with a testcompound.

(3) Quantification of the Protein or Partial Peptide of the Invention orSalts Thereof

The antibody against the protein etc. of the invention (also referred tohereinafter as the antibody of the invention) can specifically recognizethe protein etc. of the invention, and thus the antibody of theinvention can be used for e.g. quantification of the protein etc. of theinvention in a test sample, particularly for quantification thereof bysandwich immunoassays.

That is, this invention provides:

(i) A method of quantifying the protein etc. of the invention in a testsolution, which comprises allowing a test solution and the labeledprotein etc. of the invention to react competitively with the antibodyof the invention and determining the proportion of the labeled proteinetc. of the invention bound to said antibody; and

(ii) A method of quantifying the protein etc. of the invention in a testsolution, which comprises allowing a test solution to react with theantibody of the invention insolubilized on a carrier and another labeledantibody of the invention simultaneously or successively and thenmeasuring the activity of the label on the insolubilizing carrier.

In the quantification method (ii) above, it is desired that one antibodyis an antibody recognizing an N-terminal region of the protein etc. ofthis invention, and the other antibody is an antibody recognizing aC-terminal region of the protein etc. of the invention.

The monoclonal antibody against the protein etc. of the invention(hereinafter, also referred to as the monoclonal antibody of theinvention) can be used not only for quantifying the protein etc. of theinvention but also for detection thereof by tissue staining etc. Forthese purposes, the antibody molecule itself or an F(ab′)₂, Fab′ or Fabfraction of the antibody molecule may be used.

The method of quantifying the protein etc. of the invention by use ofthe antibody of the invention is not particularly limited as long as itis a method wherein the amount of the antibody, the antigen or anantigen-antibody conjugate, corresponding to the amount of the antigen(e.g., the amount of the protein) in a test solution, is detected bychemical or physical means and calculated on the basis of a standardcurve prepared using standard solutions containing known amounts of theantigen. For example, nephelometry, the competitive method, theimmunometric method and the sandwich method are preferably used, amongwhich the sandwich method described later is particularly preferablyused in respect of sensitivity and specificity.

As the label used in the measurement method using the labeled material,for example a radioisotope, an enzyme, a fluorescent material or aluminescent material is used. The radioisotope includes, for example,[¹²⁵I], [¹³¹I], [³H], [¹⁴C] etc. The enzyme is preferably a stableenzyme with high specific activity, which includes, for example,β-galactosidase, β-glucosidase, alkali phosphatase, peroxidase andmalate dehydrogenase. The fluorescent material includes, for example,fluorescamine and fluorescein isocyanate. The luminescent materialincludes, for example, luminol, luminol derivatives, luciferin, andlucigenin. Further, a biotin-avidin system can also be used for bindingthe label to the antibody or antigen.

The antigen or antibody may be insolubilized by physical adsorption orvia chemical bonding used conventionally for insolubilization orimmobilization of proteins, enzymes etc. The carrier includes insolublepolysaccharides such as agarose, dextran and cellulose, synthetic resinsuch as polystyrene, polyacrylamide and silicon, and glass etc.

In the sandwich method, a test sample is allowed to react with theinsolubilized monoclonal antibody of the invention (primary reaction),then another labeled monoclonal antibody of the invention is allowed toreact therewith (secondary reaction), and the activity of the labelingagent on the insolubilizing carrier is measured, whereby the protein ofthe invention in the test sample can be quantitatively determined. Theprimary and secondary reactions may be carried out in the reverse order,simultaneously, or separately after a predetermined time. The labelingagent and the method of insolubilization may be in accordance with thosedescribed above. For the purpose of improving measurement sensitivityetc., immunoassays by the sandwich method may make use of not only onekind of antibody but also a mixture of two or more kinds of antibodiesas the solid-phase antibody or labeling antibody.

In the method of measuring the protein etc. of the invention by thesandwich method according to this invention, the monoclonal antibodiesof the invention used in the primary and secondary reactions arepreferably those antibodies to which the protein etc. of the inventionare bound at different sites. That is, when the antibody used in e.g.the secondary reaction recognizes the C-terminal region of the proteinetc. of the invention, while the other antibody used in the primaryreaction recognizes other regions (e.g. the N-terminal region) than theC-terminal region.

The monoclonal antibody of the invention can be used not only in thesandwich method but also in other measurement systems such ascompetitive method, immunometric method and nephelometry.

In the competitive method, an antigen in a test sample and its labeledantigen are allowed to react competitively with the antibody, then theunreacted labeled antigen (F) and the labeled antibody (B) bound to theantibody are separated from each other (B/F separation), and the amountof either labeled B or F is determined to quantify the amount of theantigen in the test sample. This reaction method employs either aliquid-phase method in which a soluble antibody is used as the antibody,and polyethylene glycol and a second antibody different from the aboveantibody are used in B/F separation, or a solid-phase method in which asolid-phase antibody is used as the primary antibody, or a solubleantibody is used as the primary antibody while a solid-phase antibody isused as the secondary antibody.

In the immunometric method, the antigen in a test solution and thesolid-phase antigen are allowed to react competitively with apredetermined amount of the labeled antibody, followed by separating thesolid phase from the liquid phase, or alternatively the antigen in atest solution is allowed to react with an excess of the labeledantibody, and then the solid-phase antigen is added thereto to permitthe unreacted labeled antibody to be bound to the solid phase, followedby separating the solid phase from the liquid phase. Then, the amount ofthe label in either phase is measured to quantify the amount of theantigen in the test solution.

In nephelometry, the amount of insoluble precipitates in gel orsolution, formed by antigen-antibody reaction, is measured. Lasernephelometry using laser scattering can be applied preferably to thecase where precipitates are obtained in a small amount because of a verysmall amount of the antigen in a test solution.

For application of these immunoassays to the quantification method ofthe invention, it is not necessary to establish special conditions,procedures etc. A measurement system for the protein etc. of theinvention can be established in an ordinary manner by those skilled inthe art by modifying the conventional conditions and procedures. Thedetails of these general technical means can be referred to in generalremarks, books etc.

For example, reference can be made to “Radioimmunoassays” edited byHirhoshi Irie (published by Kodansha in 1974), “Radioimmunoassays” (2ndedition) edited by Hiroshi Irie (published by Kodansha in 1979), “EnzymeImmunoassays” edited by Eiji Ishikawa (published by Igakushoin in 1978),“Enzyme Immunoassays” (2nd edition) edited by Eiji Ishikawa (publishedby Igakushoin in 1982), “Enzyme Immunoassays” (3rd edition) edited byEiji Ishikawa (published by Igakushoin in 1987), Methods in Enzymology,Vol. 70 (Immunochemical Techniques (Part A), Methods in Enzymology, Vol.73 (Immunochemical Techniques (Part B)), Methods in Enzymology, Vol. 74(Immunochemical Techniques (Part C)), Methods in Enzymology, Vol. 84(Immunochemical Techniques (Part D: Selected Immunoassays)), Methods inEnzymology, Vol. 92 (Immunochemical Techniques (Part E: MonoclonalAntibodies and General Immunoassay Methods)), and Methods in Enzymology,Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology andMonoclonal Antibodies)) (which are published by Academic Press Ltd.).

By using the antibody of the invention in the manner as described above,the protein etc. of the invention can be quantified with goodsensitivity.

When a reduction in the protein etc. of the invention is detected byquantifying the concentration of the protein etc. of the invention byusing the antibody of the invention, patients examined can be diagnosedas having various cancers (for example, uterine cancer, endometriumtumor, breast cancer, colon cancer, prostate cancer, lung cancer, kidneycancer, neuroblastoma, bladder cancer, melanoma etc.), or it ispredicted that they will probably have such diseases.

Further, the antibody of the invention can be used to detect the proteinetc. of the invention present in test samples such as body fluids andtissues. The antibody of the invention can also be used for preparationof an antibody column used in purification of the protein etc. of theinvention, for detection of the protein etc. of the invention in eachfraction during purification, and for analysis of the behavior of theprotein etc. of the invention in cells examined.

The antibody of the invention having the action of neutralizing theactivity of the protein etc. of the invention (neutralizing antibody)can be used for example as an immunosupressant or an antiinflammatoryagent not only against various diseases such as autoimmune diseases andinfectious hyper-immunoreactions after transplantation of organs andtissues.

Hereinafter, the neutralizing antibody of the invention in “(4)Pharmaceutical composition containing the antibody of the invention” isalso referred to as the antibody of the invention.

The therapeutic or preventive agent containing the antibody of theinvention against the diseases described above can be administeredorally or parenterally as a liquid agent directly or as a pharmaceuticalcomposition in a suitable form into humans or mammals (e.g., rat,rabbit, sheep, pig, cow, cat, dog, monkey etc.). The dose is varieddepending on the subject of administration, intended disease,conditions, administration route etc., but for example, the antibody ofthe invention is conveniently injected intravenously once to about 5times preferably once to about 3 times every day, in a single dose ofusually about 0.01 to 20 mg/kg, preferably about 0.1 to 10 mg/kg, morepreferably 0.1 to 5 mg/kg, to treat and prevent autoimmune diseases foradults. A similar dose can be administered in other parenteraladministration or oral administration. In the case of particularlysevere conditions, the dose may be increased depending on theconditions.

The antibody of the invention can be administered as such or as asuitable pharmaceutical composition. The pharmaceutical composition usedin the administration described above contains the above antibody orsalts thereof, pharmaceutically acceptable carriers, diluents orexcipients. The composition is provided in a pharmaceutical formsuitable for oral or parenteral administration.

That is, the composition for e.g. oral administration is in a solid orliquid form, specifically in the form of tablets (including sugar-coatedtablets and film-coated tablets), pills, granules, powders, capsules(including soft capsules), syrups, emulsions, suspensions etc. Thecomposition is produced in a method known per se and contains carriers,diluents or excipients generally used in the field of pharmaceuticalmanufacturing. For example, the carriers and excipients used in tabletsinclude lactose, starch, sucrose, magnesium stearate etc.

The composition used for parenteral administration includes e.g.injections, suppositories etc., and the injections are in pharmaceuticalforms such as intravenous injection, subcutaneous injection,intracutaneous injection, intramuscular injection and intravenousinfusion. These injections are prepared in a usual manner by dissolving,suspending or emulsifying the antibody or its salt in a germ-free,aqueous or oily solution used conventionally in injections. The aqueoussolution for injection includes e.g. physiological saline or an isotonicsolution containing glucose and other auxiliary chemicals, and may beused in combination with suitable solubilizers such as alcohols (e.g.,ethanol etc.), polyalcohols (e.g., propylene glycol, polyethyleneglycol) and non-ionic surfactants (e.g., Polysorbate 80™ and HCO-50(i.e. polyoxyethylene (50 mol) adduct of hydrogenated castor oil). Theoily solution includes e.g. sesame oil, soybean oil etc., and may beused in combination with solubilizers such as benzyl benzoate, benzylalcohol etc. The prepared injection is introduced usually into suitableampoules. The suppositories used in administration into rectum areprepared by mixing the antibody or its salt with a conventional base forsuppositories.

The oral or parenteral pharmaceutical composition described above isproduced conveniently in a unit form adapted to the prescribed dose ofthe active ingredient. The pharmaceutical composition in such a unitform includes e.g. tablets, pills, capsules, injections (ampoules) andsuppositories, where the antibody is contained in an amount of usually 5to 500 mg/dose in these pharmaceutical forms, preferably 5 to 100mg/dose in the injections or 10 to 250 mg/dose in other pharmaceuticalforms.

The respective compositions described above may contain any other activeingredients that do not generate undesired interaction uponincorporation into the antibody.

When bases or amino acids are expressed in abbreviations in thisspecification and drawings, the following abbreviations are used inaccordance with Commission on Biochemical Nomenclature IUPAC-IUB. Ifamino acids can occur as optical isomers, L-isomers are referred tounless otherwise specified.

-   DNA: deoxyribonucleic acid-   cDNA: complementary deoxyribonucleic acid-   A: adenine-   T: thymine-   G: guanine-   C: cytosine-   I: inosine-   R: adenine (A) or guanine (G)-   Y: thymine (T) or cytosine (C)-   M: adenine (A) or cytosine (C)-   K: guanine (G) or thymine (T)-   S: guanine (G) or cytosine (C)-   W: adenine (A) or thymine (T)-   B: guanine (G), guanine (G) or thymine (T)-   D: adenine (A), guanine (G) or thymine (T)-   V: adenine (A), guanine (G) or cytosine (C)-   RNA: ribonucleic acid-   mRNA: messenger ribonucleic acid-   dATP: deoxyadenosine triphosphate-   dTTP: deoxythymidine triphosphate-   dGTP: deoxyguanosine triphosphate-   dCTP: deoxycytidine triphosphate-   ATP: adenosine triphosphate-   Gly: glycine-   Ala: alanine-   Val: valine-   Leu: leucine-   Ile: isoleucine-   Ser: serine-   Thr: threonine-   Cys: cysteine-   Met: methionine-   Glu: glutamic acid-   Asp: aspartic acid-   Lys: lysine-   Arg: arginine-   His: histidine-   Phe: phenyl alanine-   Tyr: tyrosine-   Trp: tryptophan-   Pro: proline-   Asn: asparagine-   Gln: glutamine-   pGlu: pyroglutamic acid

The substituents, protective groups and reagents appearing frequently inthis specification are expressed in the following symbols.

-   Me: methyl group-   Et: ethyl group-   Bu: butyl group-   Ph: phenyl group-   TC: thiazolidine-4 (R)-carboxamide group-   Tos: p-toluene sulfonyl-   CHO: formyl-   Bzl: benzyl-   Cl₂-Bzl: 2,6-dichlorobenzyl-   MBzl: methoxybenzyl-   MeBzl: 4-methylbenzyl-   OcHex: cyclohexyl ester-   OBzl: benzyl ester-   Bom: benzyloxymethyl-   Z: benzyloxycarbonyl-   Cl—Z: 2-chlorobenzyloxycarbonyl-   Br—Z: 2-bromobenzyloxycarbonyl-   Boc: t-butoxycarbonyl-   DNP: dinitrophenyl-   Trt: trityl-   Bum: t-butoxymethyl-   Fmoc: N-9-fluorenyl methoxycarbonyl-   HOBt: 1-hydroxybenztriazole-   HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-   HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide-   DCC: N,N′-dicyclohexyl carbodiimide-   DMF: N,N-dimethylformamide-   TEA: triethylamine-   WSCD: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-   EDTA: ethylene diamine tetraacetate-   SDS: sodium dodecyl sulfate

The sequence numbers in the Sequence Listing in this specification showthe following sequences:

SEQ ID NO:1 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 1.

SEQ ID NO:2 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 1.

SEQ ID NO:3 shows the nucleotide sequence of DNA coding for thehuman-derived protein of the invention having the amino acid sequenceset forth in SEQ ID NO:4.

SEQ ID NO: 4 shows the amino acid sequence of the human-derived protein(CSP2 protein) of the invention.

SEQ ID NO:5 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 2.

SEQ ID NO:6 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 2.

SEQ ID NO:7 shows the FLAG sequence.

SEQ ID NO:8 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 6.

SEQ ID NO:9 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 6.

SEQ ID NO:10 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 6.

SEQ ID NO:11 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 7.

SEQ ID NO:12 shows the nucleotide sequence of the primer (synthetic) DNAused in Example 7.

The transformant Escherichia coli JM109/pCR2.1-CSP2 obtained in Example1 below has been deposited under FERM BP-7091 from Mar. 16, 2000 withInternational Patent Organism Depositary (IPOD), National Institute ofAdvanced Industrial Science and Technology (AIST), Chuo No. 6, Higashi1-1-1, Tsukuba City, Ibaraki Pref., JP, and under IFO 16363 from Feb.16, 2000 with Institute for Fermentation, Osaka (IFO), Juso-honmachi2-17-85, Yodogawa-ku, Osaka City, Osaka Pref., JP.

Hereinafter, this invention is described in more detail by reference tothe Examples, which are not intended to limit this invention. Thegenetic manipulation using E. coli was conducted in accordance withmethods described in Molecular Cloning.

EXAMPLE 1 Cloning of CSP2 cDNA

PCR was conducted using human kidney cDNA (Marathon-Ready™ cDNA;Clontech) as the template and two primers i.e. primer 1 (SEQ ID NO:1)and primer 2 (SEQ ID NO:2). Using Advantage 2 Polymerase Mixture(Clontech), the PCR reaction was carried out (1) at 95° C. for 1 minuteand then (2) 30 times at 95° C. for 30 seconds, 62° C. for 30 seconds,and 68° C. for 2 minutes, followed by (3) extension reaction at 68° C.for 5 minutes. After the reaction, the reaction product was cloned intoplasmid vector pCR2.1 (Invitrogen) according to a protocol of TA CloningKit (Invitrogen). As a result of analysis of each clone sequence, a cDNAsequence (SEQ ID NO:3) coding for a novel secretory protein wasobtained. An amino acid sequence (SEQ ID NO:4) deduced from this cDNAwas designated CSP2. A transformant harboring the cDNA shown in SEQ IDNO:3 was designated Escherichia coli JM109/pCR2.1-CSP2.

EXAMPLE 2 Construction of an Expression Vector for Expressing CSP2 inAnimal Cells

An expression vector for expressing CSP2 in animal cells was obtained byinserting a DNA fragment containing an open reading frame (ORF) codingfor CSP2 into an animal cell expression vector pCAN618FLAG. pCAN618FLAGis derived from plasmid vector pCAN618 (International Application; PCTJP00/05685), in which the desired protein can be expressed as a FLAGfusion protein by ligating the ORF to a nucleotide sequence coding for8-amino-acid FLAG sequence (SEQ ID NO:7;Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) present just after Sal I site and atermination codon. First, PCR was conducted using the CSP2-coding cDNAas the template and a synthetic DNA (SEQ ID NO:5) designed to have arestriction enzyme Mfe I recognition site just before a translationinitiation codon and a synthetic DNA (SEQ ID NO:6) designed to have arestriction enzyme Sal I recognition site after amino acid 222 in CSP2protein. Using Advantage 2 Polymerase Mixture (Clontech), the PCRreaction was carried out (1) at 94° C. for 1 minute and then (2) 30times at 98° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 1minute, followed by (3) extension reaction at 72° C. for 10 minutes,whereby a DNA fragment containing the ORF for CSP2 was obtained. ThisDNA fragment was cloned into plasmid vector pVR2.1-TOPO (Invitrogen)according to a protocol of TA Cloning Kit (Invitrogen). The resultantplasmid was cleaved with restriction enzymes Mfe I and Sal I, and theinserted fragment was recovered and inserted into between Eco RI/Sal Isites in pCAN618FLAG, to give an expression vector pCAN618/CSP2-FLAG forexpressing human CSP2 protein in animal cells.

EXAMPLE 3 Expression of CSP2 in COS-7 Cells

2×10⁶ COS-7 cells were cultured for 24 hours in DMEM (medium; Gibco BRL)containing 10% FBS (fetal bovine serum) in a 10-cm Petri dish. Theexpression vector pCAN618/CSP2-FLAG obtained in Example 2 or the controlvector pCAN618 was introduced by Lipofect AMINE (Gibco BRL) into thecells, and the cells were further cultured for 18 hours. Then, themedium was exchanged with Opti-MEM (medium; Gibco BRL) containing 0.05%CHAPS, and the cells were further cultured for 24 hours, and the culturesupernatant was recovered. After the suspended cells were removed bycentrifugation, 1 μl SDS-sample buffer containing 2-mercaptoethanol wasadded to 1 μl of the supernatant which was then electrophoresed on 16%Peptide-PAGE (TEFCO) and transferred electrically onto a PVDF membrane(Amersham). As the primary antibody, an anti-FLAG antibody (mouse IgG;Sigma) was used, and as the secondary antibody, HRP (horseradishperoxidase)-labeled anti-mouse IgG antibody (Amersham) was used.Coloration was performed under light exposure for 5 minutes by using anECL plus Western Blot Detection System (Amersham). The results are shownin FIG. 1. In FIG. 1, lane 2 is the expression vector(pCAN618/CSP2-FLAG), and lane 1 is the control plasmid (pCAN618). As isevident from FIG. 1, the product recognized by anti-FLAG antibody wasconfirmed in the culture supernatant.

EXAMPLE 4 Purification of CSP2 Protein from a Culture Supernatant ofCOS7 Cells

Because it was confirmed in Example 3 that CSP2 protein is secreted intothe culture supernatant of COS7 cells, CSP2-FLAG protein was purifiedfrom the culture supernatant of COS7 cells. According to the method inExample 3, the expression vector obtained in Example 2 was introducedinto COS7 cells in thirty 10-cm Petri dishes, and the culturesupernatant was recovered. The recovered culture supernatant wascentrifuged to remove the cells etc., and then adsorbed onto anti-FLAGM2-agarose affinity gel (SIGMA), and the objective protein was elutedwith FLAG peptide (SEQ ID NO:7; Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Thiseluate was dialyzed against TBS buffer (pH 7.2), concentrated to avolume of 500 μl by Centricon-10 (Amicon), to give a purifiedpreparation. This sample was subjected to SDS-PAGE and subsequent silverstaining, whereby a broad band was detected at a position of about 40kDa. Further, this band was quantified for its protein by Micro BCAProtein Assay Kit (Piercr), to give 15 μl protein.

EXAMPLE 5 Determination of an N-terminal Amino Acid Sequence of thePurified CSP2-FLAG Protein

20 μl of the purified preparation obtained in Example 4 was diluted with0.1% TFA and adsorbed onto a PVDF membrane to remove low-molecularcontaminants. The purified preparation was analyzed in PL-Prosorb cycleby a protein sequencer Procice 491 cLC (Applied Biosystems). As aresult, the amino acid residues 1. histidine (0.92 pmol), 2. serine(1.00 pmol) and 3. leucine (1.87 pmol) were obtained as major productsin this order from the N-terminal, and the amino acid residues 1.glycine (0.70 pmol), 2. histidine (0.43 pmol), 3 serine (0.64 pmol) and4. leucine (1.11 pmol) were obtained as second components in this orderfrom the N-terminal. From this result, it was found that a N-terminalsignal sequence of 30 or 29 amino acid residues is cleaved off from aprecursor protein of CSP2 protein, and the resulting protein beginningat the histidine residue 31 or the glycine residue 30 is secreted asmature CSP2 protein into the medium.

EXAMPLE 6 Expression of a Fusion Protein of CSP2 Protein with Human IgGFc Region

An expression vector for expressing CSP2 as a fusion protein with humanIgG Fc region was constructed in the following procedure. First, PCR wascarried out using human spleen IgG Fc cDNA (Clontech) as the templateand synthetic DNAs (SEQ ID NOS:8 and 9) having a restriction enzyme XhoI or Not I site as an anchor. Using Advantage 2 Polymerase Mixture(Clontech), the PCR reaction was carried out (1) at 94° C. for 1 minuteand then (2) 35 times at 96° C. for 10 seconds, 60° C. for 30 secondsand 72° C. for 1 minute, followed by (3) extension reaction at 72° C.for 10 minutes, whereby a DNA fragment coding for human IgG Fc regionwas obtained. This DNA fragment was cloned into plasmid vectorpCR2.1-TOPO (Invitrogen) according to a protocol of TA Cloning Kit(Invitrogen). The resultant plasmid was cleaved with restriction enzymesXho I and Not I, and the inserted fragment was recovered and insertedinto between Xho I/Not I sites in pCAN618, to give pCAN618Fc. Then, PCRwas carried out using the cDNA coding for CSP2 protein as the templateand the synthetic DNA (SEQ ID NO:5) and a synthetic DNA (SEQ ID NO:10)designed to have a restriction enzyme Xho I recognition site after aminoacid 221 in CSP2 protein. Using Advantage 2 Polymerase Mixture(Clontech), the PCR reaction was carried out (1) at 94° C. for 1 minuteand then (2) 25 times at 98° C. for 10 seconds, 60° C. for 30 secondsand 72° C. for 1 minute, followed by (3) extension reaction at 72° C.for 10 minutes, whereby a DNA fragment containing the ORF for CSP2 wasobtained. This DNA fragment was cleaved with restriction enzymes Mfe Iand Xho I, recovered and inserted into between Eco RI/Xho I sites inpCAN618Fc, to give the expression vector pCAN618/CSP2-Fc for expressinghuman CSP2 protein in animal cells.

The resultant expression vector pCAN618/CSP2-Fc was introduced into COS7cells in two 10-cm Petri dishes in the same manner as in Example 4, andthe culture supernatant was recovered. The recovered culture supernatantwas centrifuged to remove cells etc. and concentrated 100-fold byCentricon-10 (Amicon). Expression of the protein was confirmed byelectrophoresis, transfer onto a PVDF membrane and coloration in thesame manner as in Example 3 (the result is not shown).

EXAMPLE 7 Establishment of Human NKG2D-Expressing CHO-K1 Cell Strain

An expression vector for expressing human NKG2D was constructed in thefollowing manner. First, PCR was carried out using human spleen NKG2DcDNA (Clontech) as the template and synthetic DNAs (SEQ ID NOS: 11 and12) having a restriction enzyme Eco RI or Not I site as an anchor. UsingAdvantage 2 Polymerase Mixture (Clontech), the PCR reaction was carriedout (1) at 95° C. for 1 minute, then (2) 5 times at 95° C. for 20seconds and 72° C. for 4 minutes, (3) 5 times at 95° C. for 20 secondsand 68° C. for 4 minutes, (4) 30 times at 95° C. for 20 seconds, 64° C.for 20 seconds and 68° C. for 4 minutes, followed by (5) extensionreaction at 68° C. for 3 minutes, whereby a DNA fragment coding forhuman NKG2D was obtained. This DNA fragment was cloned into plasmidvector pCR2.1-TOPO (Invitrogen) according to a protocol of TA CloningKit (Invitrogen). The resultant plasmid was cleaved with restrictionenzymes Eco RI and Not I, and the inserted fragment was recovered andinserted into between Eco RI/Not I sites in pCAN618, to givepCAN618/hNKG2D.

The resultant expression vector pCAN618/hNKG2D was introduced intoCHO-K1 cells by using Lipofect AMINE (Gibco BRL). After theintroduction, the cells into which the expression vector had beenintroduced were selected in the presence of 0.5 mg/ml Geneticin (WakoPure Chemical Industries, Ltd.), to give CHO-K1 cell strainCHO-K1/hNKG2D-11 expressing human NKG2D.

EXAMPLE 8 Binding of CSP2-Fc Protein to the Human NKG2D-ExpressingCHO-K1 Cells

The human NKG2D-expressing CHO-K1 cell strain CHO-K1/hNKG2D-11 obtainedin Example 7 was washed twice with PBS/1% FBS and suspended in 50 μlPBS/1% FBS containing 10 μl concentrate of the culture supernatant ofthe CSP2-Fc-expressing COS7 cells obtained in Example 6. The suspensionwas reacted at 0° C. for 60 minutes for binding them, then washed twicewith 200 μl PBS/1% FBS, and suspended in 50 μl PBS/1% FBS containing 1μl anti-human IgG (Fc)-FITC conjugate (Caltag). The suspension wasreacted at 0° C. for 60 minutes for labeling, then washed twice with 200μl PBS/1% FBS, and suspended again in 600 μl PBS/1% FBS. When thesuspension was analyzed with a flow cytometer FACS Vantage (BectonDickinson), cells with high intensity of FITC fluorescence wererecognized, and the evident binding of CSP2-Fc protein to human NKG2Dexpressed on the surface of CHO-K1 cells was observed (FIG. 2A).

The binding (FIG. 2B), to CHO-K1 cells (control), of the concentrate ofthe culture supernatant of the CSP2-Fc-expressing COS7 cells obtained inExample 6, and the binding (FIG. 2C), to the above CHO-K1/hNKG2D-11, ofa concentrate of a culture supernatant of a Mock plasmid-containing COS7cells obtained by introducing a Mock plasmid into the cells in the samemanner as in Example 6 was examined in the same manner.

The CSP2-Fc bound to the cells was detected by staining withFITC-labeled anti-human IgG (Fc) antiserum and subsequent FACS.

Evident enhancement of fluorescence intensity was observed uponaddition, to the CHO-K1 cells expressing human NKG2D, of the culturesupernatant concentrate of the CSP2-Fc-expressing COS7 cells (FIG. 2A)as compared with the culture supernatant concentrate of the Mockplasmid-containing COS7 cells (FIG. 2B). When the control CHO-K1 cellsnot expressing NKG2D was used, there was no enhancement of fluorescenceintensity by CSP2-Fc (FIG. 2C). The graphs A to C are collectively shownin FIG. 2D. From these results, CSP2-Fc was revealed to bind to NKG2Dexpressed on the surface of the CHO-K1 cells, and NKG2D was found to bea receptor specific to CSP2 protein.

Industrial Applicability

The protein etc. of the invention have e.g. the activity of biding toNKG2D and the action of activating immunocytes, and are thus useful asan agent for treating and preventing various cancers (for example,uterine cancer, endometrium tumor, breast cancer, colon cancer, prostatecancer, lung cancer, kidney cancer, neuroblastoma, bladder cancer,melanoma etc.). Further, the protein of the invention is useful as areagent for screening a compound or a salt thereof promoting orinhibiting the activity of the protein of the invention, and aninhibitor obtained by screening is expected to act as animmunosupressant and an antiinflammatory agent.

Further, the antibody against the protein of the invention canspecifically recognize the protein of the invention and is thus usableto quantify the protein of the invention in a sample and usable as adiagnostic agent for the various cancers described above. Further, thehumanized antibody of the invention can be used as an immunosupressantor an antiinflammatory agent.

Sequence Listing Free Text

SEQ ID NO: 1

Designed oligonucleotide primer to amplify DNA encoding CSP2

SEQ ID NO:2

Designed oligonucleotide primer to amplify DNA encoding CSP2

SEQ ID NO:5

Designed oligonucleotide primer to amplify DNA encoding CSP2

SEQ ID NO:6

Designed oligonucleotide primer to amplify DNA encoding CSP2

SEQ ID NO:7

FLAG sequence of pCAN618FLAG

SEQ ID NO:8

Designed oligonucleotide primer to amplify DNA encoding IgG Fc

SEQ ID NO:9

Designed oligonucleotide primer to amplify DNA encoding IgG Fc

SEQ ID NO: 10

Designed oligonucleotide primer to amplify DNA encoding CSP2

SEQ ID NO:11

Designed oligonucleotide primer to amplify DNA encoding NKG2D

SEQ ID NO:12

Designed oligonucleotide primer to amplify DNA encoding NKG2d

1. An isolated protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof:
 2. A partial peptide of the protein according to claim 1, or an amide, an ester or a salt thereof.
 3. An isolated DNA comprising a DNA coding for the protein according to claim
 1. 4. The DNA according to claim 3, which comprises the nucleotide sequence set forth in SEQ ID NO:3.
 5. An isolated DNA comprising a DNA coding for the partial peptide according to claim
 2. 6. A recombinant vector comprising a DNA encoding for the protein of claim 1 or a partial peptide thereof.
 7. A transformant transformed with the recombinant vector according to claim
 6. 8. A process for producing a protein according to claim 1, the partial peptide or an amide, an ester or a salt thereof, which comprises transforming a host cell with an expressible recombinant vector comprising a nucleic acid which encodes for said protein, culturing the transformant under conditions suitable for expression of said protein, and obtaining the protein of claim 1, or the partial peptide thereof.
 9. An antibody against the protein according to claim 1 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof.
 10. A diagnostic composition comprising an DNA which encodes for the protein of claim 1, or a partial peptide thereof, or an antibody which binds specifically to a protein of claim 1 or a partial peptide thereof.
 11. A pharmaceutical composition comprising the protein according to claim 1 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof.
 12. A pharmaceutical composition according to claim 11, for preventing and treating cancers.
 13. A method of screening a compound or a salt thereof for promoting or inhibiting an activity of the protein according to claim 1 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, which comprises contacting a test compound with the protein according to claim 1 or a salt thereof, or the partial peptide thereof or an amide thereof, an ester thereof or a salt thereof in a screening assay and detecting change in activity of said protein as compared with a baseline control.
 14. A kit for screening a compound or a salt thereof promoting or inhibiting an activity of the protein according to claim 1 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, which comprises the protein according to claim 1 or a salt thereof, or the partial peptide thereof or an amide thereof, an ester thereof or a salt thereof and instructions for screening.
 15. A compound or a salt thereof promoting an activity of the protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, which is obtainable by the screening method according to claim
 13. 16. A pharmaceutical composition comprising the compound according to claim 15 or a salt thereof and a pharmaceutically acceptable carrier, diluent or excipient.
 17. A method for preventing or treating cancer in a mammal in need thereof comprising administering an effective amount of a pharmaceutical composition according to claim 16, to said mammal.
 18. A method for making a pharmaceutical composition having an anticancer action comprising combining (i) an isolated protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, or (ii) a compound or a salt thereof promoting an activity of said isolated protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, which is obtained by the screening method according to claim 13, with a pharmaceutically acceptable carrier, diluent or excipient.
 19. A method of preventing and treating cancer, which comprises administering to mammals (i) the protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, or (ii) the compound or a salt thereof promoting an activity of the protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, which is obtained by the screening method according to claim
 13. 20. A compound or a salt thereof inhibiting an activity of the protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial peptide thereof or an amide, an ester or a salt thereof, which is obtained by the screening method according to claim
 13. 21. A pharmaceutical composition comprising the compound according to claim 20 or a salt thereof and a pharmaceutically acceptable carrier, diluent or excipient.
 22. The pharmaceutical composition according to claim 21, which is an immunosupressant or an antiinflammatory composition.
 23. A method for making a pharmaceutical composition having an immunosuppressive or anti-inflammatory action comprising combining (i) a compound or a salt thereof which inhibits the activity of the a protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial thereof or an amide, an ester or a salt thereof, which is obtained by the screening method according to claim 13, or (ii) an antibody which binds specifically thereto with a pharmaceutically acceptable carrier, diluent or excipient.
 24. A method of immunorepression or a method of treating inflammations, which comprises administering to mammals the compound or a salt thereof inhibiting the activity of the protein comprising an amino acid sequence identical or substantially identical with the amino acid sequence set forth in SEQ ID NO:4 or a salt thereof, or the partial peptide thereof or an amide thereof, an ester thereof or a salt thereof, which is obtained by the screening method according to claim 13, or an antibody which binds specifically thereto. 